Sugar chain synthases

ABSTRACT

The present invention provides O-glycan α2,8-sialyltransferase which has novel substrate specificity and substrate selectivity, and β-galactoside α2,6-sialyltransferase which has novel action and substrate specificity. The sialyltransferase of the present invention can be used as a medicament for suppression of cancer metastasis, prevention of virus infection, suppression of inflammatory response, or activation of neural cells.

TECHNICAL FIELD

The present invention relates to a glycosylating enzyme and DNA encodingthe enzyme. More specifically, the present invention relates to anenzyme (O-glycan α2,8-sialyltransferase, ST8Sia VI) that efficientlytransfers sialic acid through an α2,8 linkage onto the sialic acidportion of a sugar chain having a Sia α2,3(6)Gal (Sia: sialic acid; Gal:galactose) structure at the terminus of O-glycans such as mucin, and DNAencoding the above enzyme; and an enzyme (ST6Gal II) that efficientlytransfers sialic acid through an α2,6 linkage onto the galactose portionof a sugar chain having a Galβ1,4GlcNAc (Gal: galactose; GluNAc:N-acetylglucosamine) structure at the terminus of sugar chains such asoligosaccharide, and DNA encoding the above enzyme. The O-glycanα2,8-sialyltransferase and β-galactoside α2,6-sialyltransferase of thepresent invention are useful as a medicament having effects ofsuppression of cancer metastasis, prevention of virus infection,suppression of inflammatory response or activation of neural cells, as areagent for increasing physiological action by adding sialic acid to asugar chain, or as an enzyme inhibitor.

BACKGROUND ART

Sialic acid is a substance responsible for important physiologicalactions such as cell-cell communication, cell-substrate interaction, andcell adhesion. The presence of sialic acid-containing sugar chains hasbeen known, and some of such chains are expressed in stage-specificmanner during development and differentiation, or in tissue-specificmanner. Sialic acid exists at the terminal position of the sugar chainof a glycoprotein or glycolipid. Introduction of sialic acid into thesesites is carried out emzymatically by transfer of sialic acid portionfrom CMP-Sia.

Enzymes having a function in such enzymatic introduction of sialic acid(sialic acid tranfer) belong to a member of glycosyltransferases calledsialyltransferases. So far, 18 types of sialyltransferases have beenknown with regard to mammals. These sialyltransferases are broadlydivided into 4 families (Tsuji, S. (1996) J. Biochem. 120, 1-13). Thisis to say, these 4 families are: α2,3-sialyltransferase (ST3Gal-family)that transfers sialic acid onto galactose through an α2,3 linkage;α2,6-sialyltransferase (ST6Gal-family) that transfers sialic acid ontogalactose through an α2,6 linkage; GalNAc α2,6-sialyltransferase(ST6GalNAc-family) that transfers sialic acid onto N-acetylgalactosaminethrough an α2,6 linkage; and α2,8-sialyltransferase (ST8Sia-family) thattransfers sialic acid onto sialic acid through an α2,8 linkage.

Of these, with regard to α2,8-sialyltransferase, cDNA cloning of 5 typesof the enzymes (ST8Sia I-V) have been achieved so far, and theirenzymatic properties have been elucidated (Yamamoto, A. et al. (1996) J.Neurochem. 66, 26-34; Kojima, N. et al. (1995) FEBS Lett. 360, 1-4;Yoshida, Y. et al. (1995) J. Biol. Chem. 270, 14628-14633; Yoshida, Y.et al. (1995) J. Biochem. 118, 658-664; Kono, M. et al. (1996) J. Biol.Chem. 271, 29366-29371). ST8Sia I is an enzyme for synthesizing aganglioside GD3, and ST8Sia V is also an enzyme for synthesizinggangliosides GD1c, GT1a, GQ1b, GT3, and so on. ST8Sia II and IV areenzymes for synthesizing polysialic acid on the N-glycans of a neuralcell adhesion molecule (NCAM). ST8Sia III is an enzyme for transferringsialic acid onto Siaα2,3Galβ1,4GlcNAc structures found in the N-glycansof glycoproteins and glycolipids. The preferred substrates for all ofthese enzymes are glycolipids or N-glycans. There have been only tworeports in which these enzymes exhibit activity toward O-glycans. A casewhere ST8Sia II and IV synthesize oligosialic acid/polysialic acid onO-glycans found in an isoform of NCAM, and a case where ST8Sia III actson the O-glycans of an adipocyte-specific glycoprotein AdipoQ (Suzuki,M. et al. (2000) Glycobiology 10, 1113; and Sato C, et al. (2001) J.Biol. Chem. 276, 28849-28856). Thus, the previously reportedα2,8-sialyltransferases do not generally utilize O-glycans as preferredsubstrates. The existence of α2,8-sialyltransferase which utilizes suchan O-glycans as preferred substrates has been unknown.

Moreover, so far, cDNA cloning of only one type of β-galactosideα2,6-sialyltransferase (ST6Gal I) has been achieved, and its enzymaticproperties have been elucidated (Hamamoto, T. and Tsuji, S. (2001)ST6Gal-I in Handbook of Glycosyltransferases and Related Genes(Taniguchi, N. et al. Eds.) pp. 295-300). ST6Gal I shows its activity onglycoproteins, oligosaccharides, and gangliosides, which have aGalβ1,4GlcNAc structure at the terminal position of their carbohydrates.ST6Gal I is an enzyme having broad substrate specificity, whosesubstrate can be not only the Galβ1,4GlcNAc structure, but also lactose(Galβ1,4Glc), or a Galβ1,3GlcNAc structure in some cases. If afunctional oligosaccharide is synthesized using an enzyme having widesubstrate specificity such as ST6Gal I, there is a possibility thatby-products might be generated when there are impurities in the rawmaterials, as these impurities would also serve as substrates. To solvethis problem, an enzyme having high selectivity is required in terms ofsubstrate specificity. However, so far, the enzyme having β-galactosideα2,6-sialyltransferase activity with high selectivity in terms ofsubstrate specificity has not been identified from mammals.

DISCLOSURE OF THE INVENTION

As stated above, only 5 types of α2,8-sialyltransferases have been knownso far. Main substrates for all of these enzymes are glycoproteinshaving N-glycans or glycolipids such as gangliosides. These enzymes showno activity toward glycoproteins having O-glycans, or show only alimited activity. It is the first object of the present invention toprovide a novel O-glycan α2,8-sialyltransferase showing high activitytoward O-glycans. It is also the object of the present invention toclone the cDNA encoding O-glycan α2,8-sialyltransferase, so as toprovide a DNA sequence encoding the above O-glycanα2,8-sialyltransferase and an amino acid sequence of the above enzyme.Moreover, it is also the object of the present invention to allow aportion necessary for the activity of the above O-glycanα2,8-sialyltransferase to express as a protein in a large quantity.

Furthermore, as stated above, only one type of β-galactosideα2,6-sialyltransferase (ST6Gal I) has been known in mammals. This enzymeshows activity toward glycoproteins, oligosaccharides, or gangliosides,which have a Galβ1,4GlcNAc structure at the terminal position of theircarbohydrates. ST6Gal I is an enzyme having a wide substratespecificity, whose substrate can be not only the Galβ1,4GlcNAcstructure, but also lactose (Galβ1,4Glc), or a Galβ1,3GlcNAc structurein some cases. It is the second object of the present invention toprovide a novel β-galactoside α2,6-sialyltransferase, which solves theabove problem regarding broad substrate specificity and shows highlyselective substrate specificity to a Galβ1,4GlcNAc structure onoligosaccharide, and DNA encoding the enzyme.

The present inventors have made intensive studies to achieve theabove-described objects. The present inventors have screened mouse brainand heart cDNA libraries, and have also performed PCR using cDNA derivedfrom mouse kidney as a template, so that they have succeeded in cloningthe cDNA encoding O-glycan α2,8-sialyltransferase. Moreover, using theamino acid sequence of human sialyltransferase ST6Gal I, the presentinventors have searched the expressed sequence tag (dbEST) database fora clone encoding a novel sialyltransferase showing a homology with theabove enzyme, and have obtained the EST clones of GenBank™ accessionNos. BE613250, BE612797, and BF038052. Furthermore, using theinformation on these nucleotide sequences, the present inventors havesearched both the dbEST database and the database of high throughputgenomic sequences of the human genome, and have obtained information onthe nucleotide sequences of the related EST clones and the genomesequence of this gene. Based on the above obtained nucleotide sequenceinformation, primers for the polymerase chain reaction method (PCR) wereprepared, and PCR was carried out using human colon-derived cDNA as atemplate. The obtained amplified fragment was ligated to the DNAfragment derived from the above-obtained EST clone, so as to obtain aclone encoding the entire coding region. Thereafter, it was confirmedthat a protein encoded by the above clone has the activity ofβ-galactoside α2,6-sialyltransferase. The present invention has beencompleted based on these findings.

That is to say, the present invention provides O-glycanα2,8-sialyltransferase, which is characterized in that it has thefollowing substrate specificity and substrate selectivity.

Substrate specificity: the substrates of the enzyme are glycoconjugateshaving a Siaα2,3(6)Gal structure (wherein Sia represents sialic acid andGal represents galactose) at the terminus thereof.

Substrate selectivity: the enzyme incorporates sialic acids intoO-glycans more preferentially than into glycolipids or N-glycans.

Preferably, the present invention provides O-glycanα2,8-sialyltransferase having either one of the following amino acidsequences: (1) an amino acid sequence shown in SEQ ID NO: 1 or 3; or (2)an amino acid sequence comprising a deletion, substitution, and/oraddition of one or several amino acids with respect to the amino acidsequence shown in SEQ ID NO: 1 or 3, and having O-glycanα2,8-sialyltransferase activity.

In another aspect of the present invention, the O-glycanα2,8-sialyltransferase gene encoding the above-described amino acidsequence of the O-glycan α2,8-sialyltransferase of the present inventionis provided.

Preferably, the present invention provides the O-glycanα2,8-sialyltransferase gene having any one of the following nucleotidesequences: (1) a nucleotide sequence corresponding to a portion betweennucleotide 77 and nucleotide 1270 of a nucleotide sequence shown in SEQID NO: 2; (2) a nucleotide sequence comprising a deletion, substitution,and/or addition of one or several nucleotides with respect to thenucleotide sequence corresponding to a portion between nucleotide 77 andnucleotide 1270 of the nucleotide sequence shown in SEQ ID NO: 2, andencoding a protein having O-glycan α2,8-sialyltransferase activity; (3)a nucleotide sequence corresponding to a portion between nucleotide 92and nucleotide 1285 of a nucleotide sequence shown in SEQ ID NO: 4; and(4) a nucleotide sequence comprising a deletion, substitution, and/oraddition of one or several nucleotides with respect to the nucleotidesequence corresponding to a portion between nucleotide 92 and nucleotide1285 of the nucleotide sequence shown in SEQ ID NO: 4, and encoding aprotein having O-glycan α2,8-sialyltransferase activity.

In another aspect of the present invention, the followings are provided:a recombinant vector (preferably, an expression vector) comprising theabove-described O-glycan α2,8-sialyltransferase gene of the presentinvention; a transformant transformed with the above recombinant vector;and a method for producing the enzyme of the present invention whereinthe above transformant is cultured and the enzyme of the presentinvention is collected from the culture.

In another aspect of the present invention, a protein which comprises anactive domain of O-glycan α2,8-sialyltransferase having any one of thefollowing amino acid sequences is provided: (1) an amino acid sequencecorresponding to a portion between positions 26 and 398 of the aminoacid sequence shown in SEQ ID NO: 1; (2) an amino acid sequencecomprising a deletion, substitution, and/or addition of one or severalamino acids with respect to the amino acid sequence corresponding to aportion between positions 26 and 398 of the amino acid sequence shown inSEQ ID NO: 1, and having O-glycan α2,8-sialyltransferase activity; (3)an amino acid sequence corresponding to a portion between positions 68and 398 of the amino acid sequence shown in SEQ ID NO: 3; and (4) anamino acid sequence comprising a deletion, substitution, and/or additionof one or several amino acids with respect to the amino acid sequencecorresponding to a portion between positions 68 and 398 of the aminoacid sequence shown in SEQ ID NO: 3, and having O-glycanα2,8-sialyltransferase activity.

In another aspect of the present invention, an extracellular secretoryprotein is provided, which comprises a polypeptide portion of the activedomain and a signal peptide of the O-glycan α2,8-sialyltransferase ofthe present invention, and has O-glycan α2,8-sialyltransferase activity.

In another aspect of the present invention, a gene encoding theabove-described extracellular secretory protein of the present inventionis provided.

In another aspect of the present invention, the followings are provided:a recombinant vector (preferably, an expression vector) comprising agene encoding the above-described extracellular secretory protein of thepresent invention; a transformant transformed with the above recombinantvector; and a method for producing the protein of the present inventionwherein the above transformant is cultured and the enzyme of the presentinvention is collected from the culture.

In another aspect of the present invention, a β-galactosideα2,6-sialyltransferase, which is characterized in that it has thefollowing action and substrate specificity, is provided.

(1) Action;

The enzyme transfers sialic acid through an α2,6 linkage into thegalactose portion of a sugar chain having a galactoseβ1,4N-acetylglucosamine structure at the terminus thereof.

(2) Substrate Specificity

The substrate of the enzyme is a sugar chain having a galactoseβ1,4N-acetylglucosamine structure at the terminus thereof, and lactoseand a sugar chain having a galactose β1,3N-acetylglucosamine structureat the terminus thereof are not the substrate of the enzyme.

In another aspect of the present invention, a β-galactosideα2,6-sialyltransferase having either one of the following amino acid isprovided: (1) an amino acid sequence shown in SEQ ID NO: 5 or 7; or (2)an amino acid sequence comprising a deletion, substitution, and/oraddition of one or several amino acids with respect to the amino acidsequence shown in SEQ ID NO: 5 or 7, and having β-galactosideα2,6-sialyltransferase activity.

In another aspect of the present invention, a β-galactosideα2,6-sialyltransferase gene encoding the above-described amino acidsequence of the β-galactoside α2,6-sialyltransferase of the presentinvention is provided.

In another aspect of the present invention, a β-galactosideα2,6-sialyltransferase gene having any one of the following nucleotidesequences is provided: (1) a nucleotide sequence corresponding to aportion between nucleotide 176 and nucleotide 1762 of a nucleotidesequence shown in SEQ ID NO: 6; (2) a nucleotide sequence comprising adeletion, substitution, and/or addition of one or several nucleotideswith respect to the nucleotide sequence corresponding to a portionbetween nucleotide 176 and nucleotide 1762 of the nucleotide sequenceshown in SEQ ID NO: 6, and encoding a protein having β-galactosideα2,6-sialyltransferase activity; (3) a nucleotide sequence correspondingto a portion between nucleotide 3 and nucleotide 1574 of a nucleotidesequence shown in SEQ ID NO: 8; and (4) a nucleotide sequence comprisinga deletion, substitution, and/or addition of one or several nucleotideswith respect to the nucleotide sequence corresponding to a portionbetween nucleotide 3 and nucleotide 1574 of the nucleotide sequenceshown in SEQ ID NO: 8, and encoding a protein having β-galactosideα2,6-sialyltransferase activity.

In another aspect of the present invention, a recombinant vectorcomprising the β-galactoside α2,6-sialyltransferase gene of the presentinvention is provided.

The recombinant vector of the present invention is preferably anexpression vector.

In another aspect of the present invention, a transformant transformedwith the recombinant vector of the present invention is provided.

In another aspect of the present invention, a method for producing theenzyme of the present invention is provided, wherein the transformant ofthe present invention is cultured and the enzyme of the presentinvention is collected from the culture.

In another aspect of the present invention, a protein comprising anactive domain of β-galactoside α2,6-sialyltransferase having any one ofthe following amino acid sequences is provided: (1) an amino acidsequence corresponding to a portion between positions 33 and 529 of theamino acid sequence shown in SEQ ID NO: 5; (2) an amino acid sequencecomprising a deletion, substitution, and/or addition of one or severalamino acids with respect to the amino acid sequence corresponding to aportion between positions 33 and 529 of the amino acid sequence shown inSEQ ID NO: 5, and having β-galactoside α2,6-sialyltransferase activity;(3) an amino acid sequence corresponding to a portion between positions31 and 524 of the amino acid sequence shown in SEQ ID NO: 7; and (4) anamino acid sequence comprising a deletion, substitution, and/or additionof one or several amino acids with respect to the amino acid sequencecorresponding to a portion between positions 31 and 524 of the aminoacid sequence shown in SEQ ID NO: 7, and having β-galactosideα2,6-sialyltransferase activity.

In another aspect of the present invention, an extracellular secretoryprotein is provided, which comprises a polypeptide portion of the activedomain and a signal peptide of the β-galactoside α2,6-sialyltransferaseof the present invention, and has β-galactoside α2,6-sialyltransferaseactivity.

In another aspect of the present invention, a gene encoding theabove-described protein of the present invention is provided.

In another aspect of the present invention, a recombinant vectorcomprising the above-described gene of the present invention isprovided.

The recombinant vector of the present invention is preferably anexpression vector.

In another aspect of the present invention, a transformant transformedwith the recombinant vector of the present invention is provided.

In another aspect of the present invention, a method for producing theprotein of the present invention is provided, wherein the transformantof the present invention is cultured and the protein of the presentinvention is collected from the culture.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the nucleotide sequences of ST8Sia VI cDNA of a mouse and ahuman, and the deduced amino acid sequences. A transmembrane domain isunderlined, sialyl motif L is double-underlined, and sialyl motif S isdashed-underlined. Histidine and glutamic acid, which are conserved insialyl motif VS, are boxed. Asparagine residues of the potentialN-linked glycosylation sites are overlined. FIG. 1A shows mouse ST8SiaVI, and FIG. 1B shows human ST8Sia VI.

FIG. 2 shows a comparison of amino acid sequences.

FIG. 2A shows a comparison made among the amino acid sequences of mousesialyltransferases ST8Sia I, ST8Sia V, and ST8Sia VI. The conservedamino acid residues among these sialyltransferases are boxed. Sialylmotif L is duble-underlined, and sialyl motif S is dashed-underlined.The conserved histidine and glutamic acid residues in sialyl motif VSare marked with asterisks.

FIG. 2B shows a comparison made between the amino acid sequence of mouse(m) ST8Sia VI and that of human (h) ST8Sia VI. Amino acids conservedbetween both the enzymes are boxed.

FIG. 3 shows an analysis of linkage specificity. A,[¹⁴C]-NeuAc-incorporated GM3 sialylated by the secretory recombinantprotein PA-mST8Sia VI of mouse ST8Sia VI was treated with α2,3-, andα2,6-linkage specific sialidase (NANase II) or with α2,3-, α2,6-, α2,8-,and α2,9-linkage specific sialidase (NANase III), and then the reactionproducts were analyzed by HPTLC (where a developing solvent consists ofchloroform:methanol: 0.02% CaCl₂=55:45:10) (upper panel).[¹⁴C]-NeuAc-incorporated 3′-sialyllactose sialylated by the secretoryrecombinant protein PA-hST8Sia VI of human ST8Sia VI was treated withNANase II or NANase III, and then the reaction products were analyzed byHPTLC (where a developing solvent consists of 1-propanol:ammoniawater:water=6:1:2.5) (lower panel). B, GM3 was sialylated by PA-mST8SiaVI, and the reaction product was analyzed by TLC immunostaining (lowerpanel). Lane 1, GD3 (1 μg); lane 2, GM3 (1 μg); and lane 3, the reactionproduct. The reaction product was reacted with an anti-GD3 monoclonalantibody KM641 and peroxidase-conjugated anti-mouse IgG+IgM (H+L)antibody, and then detected using an ECL system.

In FIG. 4, Fetuin was [¹⁴C]-NeuAc-incorporated by ST8Sia III or ST8SiaVI and then treated with N-glycanase. The [¹⁴C]-NeuAc-incorporatedFetuin was treated with N-glycanase, and the treated product wasanalyzed by SDS-PAGE. Thereafter, it was visualized with a BAS2000 radioimage analyzer.

FIG. 5 shows effects of the overexpression of the mouse ST8Sia VIfull-length cDNA in COS-7 cells.

FIG. 5A shows results of the TLC immunostaining using ananti-NeuAcα2,8NeuAcα2,3Gal antibody S2-566. Lane 1, standard GD3substance (0.5 μg); lane 2, standard GQ1b (0.5 μg); lane 3, an acidicglycolipid fraction extracted from control COS-7 cells (30 mg); and lane4, an acidic glycolipid fraction extracted from COS-7 cells (30 mg) intowhich a mouse full-length ST8Sia VI expression vector pRc/CMV-ST8Sia VIhad been introduced.

In FIG. 5B, microsome fractions were prepared from COS-7 cells, or COS-7cells into which pRc/CMV-ST8Sia VI has been introduced. Then they weresubjected to SDS-PAGE (45 μg/lane), and transferred to a PVDF membrane,and western blotting was performed using an S2-566 antibody. Lane 1, themicrosome fraction prepared from control COS-7 cells; lane 2, themicrosome fraction prepared from COS-7 cells into which pRc/CMV-ST8SiaVI has been introduced; lane 3, the N-glycanase-treated microsomefraction prepared from the control COS-7 cells; and lane 4, theN-glycanase-treated microsome fraction prepared from the COS-7 cellsinto which pRc/CMV-ST8Sia VI had been introduced. Asterisks are attachedto main bands which are recognized by the S2-566 antibody and aregenerated as a result of the introduction of ST8Sia VI cDNA.

FIG. 6 shows the expression patterns of mouse and human ST8Sia VI genes.

FIG. 6A shows results of the expression pattern of the mouse ST8Sia VIgene analyzed by northern blotting with poly(A)+RNA (approximately 2μg/lane) prepared from various types of mouse organs.

FIG. 6B shows results of the expression pattern of the human ST8Sia VIgene analyzed by PCR using a Multiple Tissue cDNA Panel (Clontech). Ashuman ST8Sia VI-specific primers, 5′-CCAGTGTCCCAGCCTTTTGT-3′(corresponding to nucleotides 608-627 in FIG. 1B) (SEQ ID NO: 17) and5′-TGAGTGGGGAAGCTTTGGTC-3′ (corresponding to a complementary strand ofnucleotides 1407-1426 in FIG. 1B) (SEQ ID NO: 18) were used. The size ofthe PCR amplified fragment is 819 bp.

FIG. 7 shows the nucleotide sequence of human ST6Gal II cDNA, itsdeduced amino acid sequence, and the hydropathy plot of the protein.

FIG. 7A shows the nucleotide sequence of human ST6Gal II cDNA, and itsdeduced amino acid sequence. The transmembrane domain is underlined.Sialyl motif L is double underlined, and sialyl motif S is dashedunderlined. Histidine and glutamic acid, which are conserved in sialylmotif VS, are boxed. Asparagine residues of the potential N-linkedglycosilation sites are overlined.

FIG. 7B shows the hydropathy plot of human ST6Gal II. A largehydrophobic region on the N-terminal region is predicted to be atransmembrane domain.

FIG. 8 shows the nucleotide sequence of mouse ST6Gal II cDNA, itsdeduced amino acid sequence, and the hydropathy plot of the protein.

FIG. 8A shows the nucleotide sequence of mouse ST6Gal II cDNA and itsdeduced amino acid sequence. The transmembrane domain is underlined.Sialyl motif L is double underlined, and sialyl motif S is dashedunderlined. Histidine and glutamic acid, which are conserved in sialylmotif VS, are boxed. Asparagine residues of the potential N-linkedglycosylation sites are overlined.

FIG. 8B shows the hydropathy plot of mouse ST6Gal II. A largehydrophobic region on the N-terminal region is predicted to be atransmembrane domain.

FIG. 9 shows a comparison of amino acid sequences.

FIG. 9A shows a comparison of the amino acid sequence of humansialyltransferase ST6Gal I and that of human sialyltransferase ST6GalII. The conserved amino acid residues between these enzymes are boxed.Sialyl motif L is double underlined, and sialyl motif S is dashedunderlined. The conserved histidine and glutamic acid residues in sialylmotif VS are marked with asterisks.

FIG. 9B shows a comparison of the amino acid sequence of human (h)ST6Gal II and that of mouse (m) ST6Gal II. The conserved amino acidresidues between these enzymes are boxed.

FIG. 10 shows the activity toward oligosaccharides. The enzyme reactionwas carried out using various oligosaccharides as substrates (10μg/lane). The figure shows the reaction product analyzed by HPTLC (wherea developing solvent consists of 1-propanol:ammoniawater:water=6:1:2.5).

FIG. 11 shows an analysis of linkage specificity. A,[¹⁴C]-NeuAc-incorporated Galβ1,4GlcNAc sialylated by human ST6Gal I(upper panel), human ST6Gal II (middle panel), and mouse ST6Gal II(lower panel) (lane 1) was treated with α2,3-linkage specific sialidase(NANase I, lane 2) or with α2,3-, and α2,6-linkage specific sialidase(NANase II, lane 3), and then the reaction products were analyzed byHPTLC (where a developing solvent consists of 1-propanol:ammoniawater:water=6:1:2.5). B, [¹⁴C]-NeuAc-incorporated Galβ1,4GlcNAcsialylated by human ST6Gal I (upper panel), human ST6Gal II (middlepanel), and mouse ST6Gal II (lower panel) (lane 1) was treated withβ-galactosidase (lane 2). As a control, Galβ1,4GlcNAc was treated withβ-galactosidase, and then an enzyme reaction was performed (lane 3).These were analyzed by HPTLC (where a developing solvent consists of1-propanol:ammonia water:water=6:1:2.5). The broad bands in lane 2 werecaused by the effects of high concentration of ammonium sulfate in theβ-galactosidase solution.

FIG. 12 shows analysis of the expression patterns of human ST6Gal I andST6Gal II genes, and a mouse ST6Gal II gene. Using human ST6Gal I- andST6Gal II-specific primers and a Multiple tissue cDNA panel (Clontech)of human tissues (A) or human tumor cells (B), the expression patternsof both genes were analyzed by PCR. One PCR cycle consists of 94° C. for1 minute, 50° C. for 1 minute, and 72° C. for 1.5 minutes. 25 cycles ofPCR was performed for the glyceraldehyde 3-phosphate dehydrogenase(G3PDH) gene, and 40 cycles of PCR was performed for human ST6Gal I andST6Gal II genes. The reaction products were analyzed by agarose gelelectrophoresis. “Sk. Muscle” means skeletal muscle, and “P. bl.Leukocyte” means peripheral blood leukocyte. FIG. 12C shows theexpression pattern of the mouse ST6Gal II gene analyzed by PCR usingmouse ST6Gal II-specific primers and the Multiple tissue cDNA panel(Clontech) of mouse tissues.

BEST MODE FOR CARRYING OUT THE INVENTION

The embodiments of the present invention and the methods for carryingout the present invention will be described in detail below.

(1) Enzyme and Protein of the Present Invention

The O-glycan α2,8-sialyltransferase of the present invention ischaracterized in that it has the following substrate specificity andsubstrate selectivity.

Substrate specificity: the substrates of the enzyme are glycoconjugateshaving a Siaα2,3(6)Gal structure (wherein Sia represents sialic acid andGal represents galactose) at the terminus thereof.

Substrate selectivity: the enzyme incorporates sialic acids intoO-glycan more preferentially than into glycolipids or N-glycans.

The above-described substrate specificity and substrate selectivity arecharacteristics which have been demonstrated by mouse- and human-derivedO-glycan α2,8-sialyltransferases obtained in examples described in thepresent specification. The O-glycan α2,8-sialyltransferase of thepresent invention is not only derived from a mouse and a human, and itis easily understandable for a person skilled in the art that the sametype of O-glycan α2,8-sialyltransferase exists in the tissues of othermammals and that those O-glycan α2,8-sialyltransferases have a highhomology to one another.

Such O-glycan α2,8-sialyltransferases are characterized in that theyhave the above-described substrate specificity and substrateselectivity. These enzymes are also included in the scope of the presentinvention.

Examples of such an O-glycan α2,8-sialyltransferase may include naturalenzymes derived from mammalian tissues and mutants thereof, andextracellular secretory proteins catalyzing the transfer of sialic acidto O-glycans through an α2,8-linkage, which are produced by geneticrecombination, such as those produced in examples described later. Theseare also included in the scope of the present invention.

O-glycan α2,8-sialyltransferase having either one of the following aminoacid sequences may be one example of the O-glycan α2,8-sialyltransferaseof the present invention: (1) an amino acid sequence shown in SEQ ID NO:1 or 3; or (2) an amino acid sequence comprising a deletion,substitution, and/or addition of one or several amino acids with respectto the amino acid sequence shown in SEQ ID NO: 1 or 3, and havingO-glycan α2,8-sialyltransferase activity.

In addition, it is to be understood that an active domain of theO-glycan α2,8-sialyltransferase of the present invention and proteinshaving O-glycan α2,8-sialyltransferase activity obtained by alterationor modification of a portion of the amino acid sequence thereof are allincluded in the scope of the present invention. Preferred examples ofsuch an active domain may include an active domain of O-glycanα2,8-sialyltransferase corresponding to a portion between positions 26and 398 of the amino acid sequence shown in SEQ ID NO: 1 and an activedomain of O-glycan α2,8-sialyltransferase corresponding to a portionbetween positions 68 and 398 of the amino acid sequence shown in SEQ IDNO: 3. A sequence portion between positions 26 and approximately 100 ofthe amino acid sequence shown in SEQ ID NO: 1 or 3 is a region calledstem, and it is considered that this region is not necessarily requiredfor the activity. Accordingly, a region corresponding to positions 101to 398 of the amino acid sequence shown in SEQ ID NO: 1 or 3 may be usedas an active domain of O-glycan α2,8-sialyltransferase.

That is to say, the present invention provides a protein which comprisesan active domain of O-glycan α2,8-sialyltransferase having any one ofthe following amino acid sequences: (1) an amino acid sequencecorresponding to a portion between positions 26 and 398 of the aminoacid sequence shown in SEQ ID NO: 1; (2) an amino acid sequencecomprising a deletion, substitution, and/or addition of one or severalamino acids with respect to the amino acid sequence corresponding to aportion between positions 26 and 398 of the amino acid sequence shown inSEQ ID NO: 1, and having O-glycan α2,8-sialyltransferase activity. (3)an amino acid sequence corresponding to a portion between positions 68and 398 of the amino acid sequence shown in SEQ ID NO: 3; and (4) anamino acid sequence comprising a deletion, substitution, and/or additionof one or several amino acids with respect to the amino acid sequencecorresponding to a portion between positions 68 and 398 of the aminoacid sequence shown in SEQ ID NO: 3, and having O-glycanα2,8-sialyltransferase activity.

On the other hand, the β-galactoside α2,6-sialyltransferase of thepresent invention is characterized in that it has the following actionand substrate specificity.

(1) Action

The enzyme transfers sialic acid through an α2,6 linkage into thegalactose portion of a sugar chain having a galactoseβ1,4N-acetylglucosamine structure at the terminus thereof.

(2) Substrate Specificity

The substrate of the enzyme is a sugar chain having a galactoseβ1,4N-acetylglucosamine structure at the terminus thereof, and lactoseand a sugar chain having a galactose β1,3N-acetylglucosamine structureat the terminus thereof are not the substrate of the enzyme.

The above-described action and substrate specifity are characteristicswhich have been demonstrated by mouse- and human-derived β-galactosideα2,6-sialyltransferases obtained in examples described in the presentspecification. The β-galactoside α2,6-sialyltransferase of the presentinvention is not only derived from a mouse and a human, but it is easilyunderstood for a person skilled in the art that the same type ofβ-galactoside α2,6-sialyltransferase exists in the tissues of othermammals and that those β-galactoside α2,6-sialyltransferases have a highhomology to one another.

Such β-galactoside α2,6-sialyltransferases are characterized in thatthey have the above-described action and substrate specifity. Theseenzymes are also included in the scope of the present invention.

Examples of such a β-galactoside α2,6-sialyltransferase may includenatural enzymes derived from mammalian tissues and mutants thereof, andextracellular secretory proteins catalyzing the transfer of sialic acidto β-galactosides through an α2,6-linkage, which are produced by geneticrecombination. These are also included in the scope of the presentinvention.

β-galactoside α2,6-sialyltransferase having either one of the followingamino acid sequences may be one example of the β-galactosideα2,6-sialyltransferase of the present invention: (1) an amino acidsequence shown in SEQ ID NO: 5 or 7; or (2) an amino acid sequencecomprising a deletion, substitution, and/or addition of one or severalamino acids with respect to the amino acid sequence shown in SEQ ID NO:5 or 7, and having β-galactoside α2,6-sialyltransferase activity.

In addition, it is to be understood that an active domain of theβ-galactoside α2,6-sialyltransferase of the present invention andproteins having β-galactoside α2,6-sialyltransferase activity obtainedby alteration or modification of a portion of the amino acid sequencethereof are all included in the scope of the present invention. Apreferred example of such an active domain may be an active domain ofβ-galactoside α2,6-sialyltransferase corresponding to a portion betweenpositions 33 and 529 of the amino acid sequence shown in SEQ ID NO: 5. Asequence portion between positions 31 and approximately 200 of the aminoacid sequence shown in SEQ ID NO: 5 is a region called stem, and it isconsidered that this region is not necessarily required for theactivity. Accordingly, a region corresponding to positions 201 to 529 ofthe amino acid sequence shown in SEQ ID NO: 1 may be used as an activedomain of β-galactoside α2,6-sialyltransferase.

Likewise, another preferred example of such an active domain may be anactive domain of β-galactoside α2,6-sialyltransferase corresponding to aportion between positions 31 and 524 of the amino acid sequence shown inSEQ ID NO: 7. A sequence portion between positions 31 and approximately200 of the amino acid sequence shown in SEQ ID NO: 7 is a region calledstem, and it is considered that this region is not necessarily requiredfor the activity. Accordingly, a region corresponding to positions * 201to 524 of the amino acid sequence shown in SEQ ID NO: 7 may be used asan active domain of β-galactoside α2,6-sialyltransferase.

That is to say, the present invention provides a protein which comprisesan active domain of β-galactoside α2,6-sialyltransferase having any oneof the amino acid sequences described below.

In another aspect of the present invention, a protein which comprises anactive domain of β-galactoside α2,6-sialyltransferase having any one ofamino acid sequences described below is provided: (1) an amino acidsequence corresponding to a portion between positions 33 and 529 of theamino acid sequence shown in SEQ ID NO: 5; (2) an amino acid sequencecomprising a deletion, substitution, and/or addition of one or severalamino acids with respect to the amino acid sequence corresponding to aportion between positions 33 and 529 of the amino acid sequence shown inSEQ ID NO: 5, and having β-galactoside α2,6-sialyltransferase activity;(3) an amino acid sequence corresponding to a portion between positions31 and 524 of the amino acid sequence shown in SEQ ID NO: 7, and (4) anamino acid sequence comprising a deletion, substitution, and/or additionof one or several amino acids with respect to the amino acid sequencecorresponding to a portion between positions 31 and 524 of the aminoacid sequence shown in SEQ ID NO: 7, and having β-galactosideα2,6-sialyltransferase activity.

In the present specification, the range of “one or several” in theexpression “an amino acid sequence comprising a deletion, substitution,and/or addition of one or several amino acids” is not particularlylimited. For example, it means 1 to 20 amino acids, preferably 1 to 10amino acids, more preferably 1 to 7 amino acids, further more preferably1 to 5 amino acids, and particularly preferably 1 to 3 amino acids.

A method for obtaining the enzyme or protein of the present invention isnot particularly limited. The protein of the present invention may be aprotein synthesized by chemical synthesis, or recombinant proteinproduced by genetic recombination.

When a recombinant protein is produced, first, DNA encoding the proteinis required to be obtained. Suitable primers are designed based on theinformation regarding amino acid sequences and nucleotide sequencesshown in SEQ ID NOS: 1 to 8 of the sequence listing in the presentspecification. Thereafter, using the obtained primers, PCR is carriedout with a suitable cDNA library as a template, so as to obtain DNAencoding the enzyme of the present invention.

For example, methods for isolating cDNA encoding O-glycanα2,8-sialyltransferases having amino acid sequences shown in SEQ ID NOS:1 and 3, and cDNA encoding β-galactoside α2,6-sialyltransferases havingamino acid sequences shown in SEQ ID NOS: 5 and 7 are described indetail in examples described later. However, a method for isolating cDNAencoding the O-glycan α2,8-sialyltransferase or β-galactosideα2,6-sialyltransferase of the present invention is not limited thereto.A person skilled in the art could easily isolate cDNA of interest byreferring to the methods described in examples below and appropriatelymodifying or altering them.

Moreover, when a partial fragment of DNA encoding the enzyme of thepresent invention is produced by the above-described PCR, the producedDNA fragments can be successively ligated to one another, so as toobtain DNA encoding a desired enzyme. The obtained DNA can be thenintroduced into a suitable expression system, so as to generate theenzyme of the present invention. Expression of the enzyme in such anexpression system will be described later in the specification.

An extracellular secretory protein, which comprises a polypeptideportion of the active domain of the O-glycan α2,8-sialyltransferase orβ-galactoside α2,6-sialyltransferase of the present invention and asignal peptide, and has O-glycan α2,8-sialyltransferase activity orβ-galactoside α2,6-sialyltransferase activity is also included in thepresent invention.

In some cases, the O-glycan α2,8-sialyltransferase and β-galactosideα2,6-sialyltransferase of the present invention may remain in cellsafter the expression and may not be secreted outside of the cells. Inaddition, there is a possibility that the production of the enzymes maybe decreased when the intracellular concentration thereof exceeds acertain limit. In order to effectively use the activity of the aboveO-glycan α2,8-sialyltransferase to transfer sialic acid to O-glycansthrough an α2,8-linkage and the activity of the above β-galactosideβ2,6-sialyltransferase to transfer sialic acid to β-galactosides throughan α2,6-linkage, a soluble form of proteins retaining the activities ofthe present enzymes and being secreted from cells during the expressionmay be produced. An example of such a protein may be an extracellularsecretory protein, which comprises a signal peptide and a polypeptideportion of the active domain of O-glycan α2,8-sialyltransferase orβ-galactoside α2,6-sialyltransferase which is involved in the activityof the O-glycan α2,8-sialyltransferase or β-galactosideα2,6-sialyltransferase of the present invention, and catalyzes thetransfer of sialic acid to O-glycans through an α2,8-linkage or toβ-galactosides through an α2,6-linkage. For example, a fusion proteinwith a signal peptide of mouse immunoglobulin IgM or protein A ispreferred embodiments of the secretory protein of the present invention.

Sialyltransferases that have been cloned so far have a domain structuresimilar to that of other glycosyltransferases. This is to say, thepreviously cloned sialyltransferases comprise an NH₂-terminal shortcytoplasmic tail, a hydrophobic signal anchor domain, a stem regionhaving proteolytic sensitivity, and a COOH-terminal large active domain(Paulson, J. C. and Colley, K. J., J. Biol. Chem., 264, 17615-17618,1989). In order to examine the position of a transmembrane domain of theO-glycan α2,8-sialyltransferase or β-galactoside α2,6-sialyltransferaseof the present invention, a hydropathy plot prepared according to themethod of Kyte and Doolittle (Kyte, J. and Doolittle, R. F., J. Mol.Biol., 157, 105-132, 1982) can be used. Moreover, in order to estimatean active domain portion, recombinant plasmids into which various typesof fragments are introduced are produced and used. An example of suchmethods is described in detail, for example, in PCT/JP94/02182. However,a method for confirming the position of a transmembrane domain orestimating an active domain portion is not limited thereto.

In order to produce an extracellular secretory protein which comprises apolypeptide portion of the active domain of O-glycanα2,8-sialyltransferase or β-galactoside α2,6-sialyltransferase and asignal peptide, for example, a sequence corresponding to the activedomain of O-glycan α2,8-sialyltransferase or β-galactosideα2,6-sialyltransferase may be subjected to inframe fusion with animmunoglobulin signal peptide sequence as a signal peptide. As such amethod, the method of Jobling (Jobling, S. A. and Gehrke, L., Nature(Lond.), 325, 622-625, 1987), for example, can be used. Further, as isdescribed in detail in examples of the present specification, a fusionprotein with a signal peptide of mouse immunoglobulin IgM or protein Amay also be produced. However, the type of a signal peptide, the methodof the fusion of a signal peptide with an active domain, and the methodof solubilization are not limited to those described above. A personskilled in the art may appropriately select a polypeptide portion whichis an active domain of O-glycan α2,8-sialyltransferase or β-galactosideα2,6-sialyltransferase, and may fuse the selected polypeptide portionwith any available signal peptide by a suitable method, so as to producean extracellular secretory protein.

(2) Gene of the Present Invention

The present invention provides a gene encoding the amino acid sequenceof the O-glycan α2,8-sialyltransferase of the present invention, and agene encoding the amino acid sequence of the β-galactosideα2,6-sialyltransferase of the present invention.

Specific examples of a gene encoding the amino acid sequence of theO-glycan α2,8-sialyltransferase of the present invention may includegenes having any one of the following nucleotide sequences: (1) anucleotide sequence corresponding to a portion between nucleotide 77 andnucleotide 1270 of a nucleotide sequence shown in SEQ ID NO: 2; (2) anucleotide sequence comprising a deletion, substitution, and/or additionof one or several nucleotides with respect to the nucleotide sequencecorresponding to a portion between nucleotide 77 and nucleotide 1270 ofthe nucleotide sequence shown in SEQ ID NO: 2, and encoding a proteinhaving O-glycan α2,8-sialyltransferase activity; (3) a nucleotidesequence corresponding to a portion between nucleotide 92 and nucleotide1285 of a nucleotide sequence shown in SEQ ID NO: 4; and (4) anucleotide sequence comprising a deletion, substitution, and/or additionof one or several nucleotides with respect to the nucleotide sequencecorresponding to a portion c between nucleotide 92 and nucleotide 1285of the nucleotide sequence shown in SEQ ID NO: 4, and encoding a proteinhaving O-glycan α2,8-sialyltransferase activity.

Specific examples of a gene encoding the amino acid sequence of theβ-galactoside α2,6-sialyltransferase of the present invention mayinclude genes having any one of the following nucleotide sequences: (1)a nucleotide sequence corresponding to a portion between nucleotide 176and nucleotide 1762 of a nucleotide sequence shown in SEQ ID NO: 6; (2)a nucleotide sequence comprising a deletion, substitution, and/oraddition of one or several nucleotides with respect to the nucleotidesequence corresponding to a portion between nucleotide 176 andnucleotide 1762 of the nucleotide sequence shown in SEQ ID NO: 6, andencoding a protein having β-galactoside α2,6-sialyltransferase activity;(3) a nucleotide sequence corresponding to a portion between nucleotide3 and nucleotide 1574 of a nucleotide sequence shown in SEQ ID NO: 8;and (4) a nucleotide sequence comprising a deletion, substitution,and/or addition of one or several nucleotides with respect to thenucleotide sequence corresponding to a portion between nucleotide 3 andnucleotide 1574 of the nucleotide sequence shown in SEQ ID NO: 8, andencoding a protein having β-galactoside α2,6-sialyltransferase activity.

The range of “one or several” in the expression “a nucleotide sequencecomprising a deletion, substitution, and/or addition of one or severalnucleotides” in the present specification is not particularly limited.For example, it means 1 to 60 nucleotides, preferably 1 to 30nucleotides, more preferably 1 to 20 nucleotides, further morepreferably 1 to 10 nucleotides, further more preferably 1 to 5nucleotides, and particularly preferably 1 to 3 nucleotides.

A gene encoding a protein comprising an active domain of the O-glycanα2,8-sialyltransferase or β-galactoside α2,6-sialyltransferase of thepresent invention, and a gene encoding an extracellular secretoryprotein which comprises a polypeptide portion which is the above activedomain and a signal peptide and has O-glycan α2,8-sialyltransferaseactivity or β-galactoside α2,6-sialyltransferase activity, are alsoincluded in the scope of the present invention.

The gene of the present invention can be obtained by the above-describedmethod.

A method of introducing a desired mutation into a certain nucleic acidsequence is known to those skilled in the art. For example, knowntechniques such as site-directed mutagenesis, PCR using degeneratedoligonucleotides, or exposure of cells containing nucleic acid to amutagenic agent or radioactive ray are used as appropriate, whereby DNAcomprising a mutation can be constructed. Such known techniques aredescribed, for example, in Molecular Cloning: A laboratory Manual,2^(nd) Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,1989; and Current Protocols in Molecular Biology, Supplements 1 to 38,John Wiley & Sons (1987-1997).

(3) Recombinant Vector of the Present Invention

The gene of the present invention can be inserted into a suitable vectorand used. The type of a vector used in the present invention is notparticularly limited. For example, it may be autonomously replicatingvector (e.g., a plasmid, etc.), or it may be a vector which isincorporated into the genome in host cells when it is introduced intothe host cells, and replicates with an incorporated chromosome.

The vector used in the present invention is preferably an expressionvector. In an expression vector, elements necessary for transcription(e.g., a promoter, etc.) are functionally ligated to the gene of thepresent invention. A promoter is a DNA sequence having transcriptionactivity in host cells, and it can appropriately be selected dependingon the type of host cells.

Examples of a promoter capable of functioning in bacterial cells mayinclude a Bacillus stearothermophilus maltogenic amylase gene promoter,a Bacillus licheniformis alpha-amylase gene promoter, a Bacillusamyloliquefaciens BAN amylase gene promoter, a Bacillus subtilisalkaline protease gene promoter, a Bacillus pumilus xylosidase genepromoter, a phage λ P_(R) or P_(L) promoter, and an Escherichia colilac, trp, or lac promoter.

Examples of a promoter capable of functioning in mammalian cells mayinclude an SV40 promoter, an MT-1 (metallothionein gene) promoter, andan adenovirus 2 major late promoter. Examples of a promoter capable offunctioning in insect cells may include a polyhedrin promoter, a P10promoter, an Autographa californica polyhedrosis basic protein promoter,a baculovirus immediate early gene 1 promoter, and a baculovirus 39Kdelayed-early gene promoter. Examples of a promoter capable offunctioning in yeast host cells may include a promoter derived from ayeast glycolytic system gene, an alcohol dehydrogenase gene promoter, aTPI1 promoter, and an ADH2-4c promoter.

Examples of a promoter capable of functioning in filamentous cells mayinclude an ADH3 promoter and a tpiA promoter.

The DNA of the present invention may be functionally ligated to a humangrowth hormone terminator, or in the case where a host is Mycomycete,the DNA may be functionally ligated to an appropriate terminator such asa TPI1 terminator or ADH3 terminator, as necessary. The recombinantvector of the present invention may also comprise elements such as apolyadenylation signal (e.g., those derived from SV40 or adenovirus 5E1bregion), a transcription enhancer sequence (e.g., SV40 enhancer), and atranslation enhancer sequence (e.g., those encoding adenovirus VA RNA).

The recombinant vector of the present invention may further comprise aDNA sequence enabling the vector to replicate in host cells. An examplemay include an SV40 replication origin (when the host cells aremammalian cells).

The recombinant vector of the present invention may further comprise aselective marker. Examples of a selective marker may include genes whosecomplements are deficient in host cells, such as dihydrofolate reductase(DHFR) or a Schizosaccharomyces pombe TPI gene, and drug resistant genesthat are resistant to ampicillin, kanamycin, tetracycline,chloramphenicol, neomycin, hygromycin, etc.

A method of ligating the DNA of the present invention, a promoter, and aterminator and/or a secretory signal sequence, as desired, to oneanother, and inserting them into a suitable vector has been well knownto those skilled in the art.

(4) Transformant of the Present Invention, and Production of Proteinusing the Same

The DNA or recombinant vector of the present invention can be introducedinto a suitable host, so as to prepare a transformant.

Any cells may be used as host cells into which the DNA or recombinantvector of the present invention is introduced, as long as they allow theDNA construct of the present invention to express therein. Examples ofhost cells may include bacteria, yeasts, Mycomycetes, and highereukaryotes.

Examples of bacterial cells may include Gram-positive bacteria such asBacillus or Streptomyces, and Gram-negative bacteria such as Escherichiacoli. Transformation of these bacteria may be carried out by theprotoplast method or known methods, using competent cells.

Examples of mammalian cells may include HEK293 cells, HeLa cells, COScells, BHK cells, CHL cells, and CHO cells. A method of transformingmammalian cells and allowing a DNA sequence introduced into the cells toexpress therein has also been known. Examples of such a method mayinclude the electroporation, the calcium phosphate method, and thelipofection method.

Examples of yeast cells may include cells belonging to Saccharomyces orSchizosaccharomyces. Examples of such cells may include Saccharomycescerevisiae and Saccharomyces kluyveri. Examples of a method ofintroducing a recombinant vector into a yeast host may include theelectroporation, the spheroplast method, and the lithium acetate method.

Examples of other fungal cells may include cells belonging tofilamentous fungi such as Aspergillus, Neurospora, Fusarium, orTrichoderma. When filamentous fungi are used as host cells,transformation can be carried out by incorporating a DNA construct intoa host chromosome to obtain recombinant host cells. Such a DNA constructcan be incorporated into a host chromosome according to known methodssuch as homologous recombination or heterologous recombination.

When insect cells are used as host cells, a recombinant gene-introducedvector and baculovirus are co-introduced into insect cells, andrecombinant virus is obtained in the culture supernatant of the insectcells. Thereafter, insect cells are infected with the recombinant virus,so that a protein is expressed (which is described in e.g. BaculovirusExpression Vectors, A Laboratory Manual; and Current Protocols inMolecular Biology, Bio/Technology, 6, 47 (1998)).

As an example of baculovirus, Autographa californica nuclearpolyhedrosis virus infecting Mamestra insects can be used.

Examples of insect cells used herein may include Spodoptera frugiperdaovarian cells Sf 9 and Sf21 [Baculovirus Expression Vectors, ALaboratory Manual, W. H. Freeman and Company, New York (1992)], andTrichoplusia ni ovarian cells HiFive (manufactured by Invitrogen).

Examples of a method of co-introducing a recombinant gene-introducedvector and the above baculovirus into insect cells to preparerecombinant virus may include the calcium phosphate method and thelipofection method.

The above transformant is cultured in a nutrient medium under conditionsenabling the expression of the introduced DNA construct. In order toisolate and purify the enzyme of the present invention from the cultureof the transformant, common protein isolation and purification methodsmay be applied.

For example, where the enzyme of the present invention is expressed in astate where it is dissolved in cells, the cells are recovered bycentrifugation after completion of the culture, and they are thensuspended in a water-type buffer solution. Thereafter, the cells weredisintegrated with an ultrasonic disintegrator or the like, so as toobtain a cell-free extract. A purified sample can be obtained from asupernatant obtained by centrifuging the above cell-free extract, usingsingly or in combination the following common protein isolation andpurification methods: solvent extraction method, salting-out usingammonium sulfate or the like, desalting, precipitation method usingorganic solvents, anion exchange chromatography using resin such asdiethylaminoethyl (DEAE) sepharose, cation exchange chromatography usingresin such as S-Sepharose FF (manufactured by Pharmacia), hydrophobicchromatography using resin such as butyl sepharose or phenyl sepharose,gel filtration using a molecular sieve, affinity chromatography,chromatofocusing, electrophoresis such as isoelectric focusing, etc.

The present invention will be further specifically described in thefollowing examples. However, these examples are not intended to limitthe scope of the present invention.

EXAMPLES Example 1 O-glycan α2,8-sialyltransferase

The following reagents and samples were used in specific examples of thepresent invention. Fetuin, asialofetuin, bovine submaxillary mucin(BSM), α1-acid glycoprotein, ovomucoid, lactosyl ceramide (LacCer), GM3,GM1a, GD1a, GD1b, GT1b, CMP-NeuAc, 6′-sialyllactose,3′-sialyl-N-acetyllactosamine, and Triton CF-54 were purchased fromSigma. 3′-sialyllactose and 6′-sialyl-N-acetyllactosamine were purchasedfrom Calbiochem. N-acetylneuraminic acid (NeuAc), GM4, Gal, andN-acetylgalactosamine (GalNAc) were purchased from Wako Pure ChemicalIndustries, Ltd. GD3 was purchased from Snow Brand Milk Products Co.,Ltd. GQ1b was purchased from Alexis Biochemicals. CMP-[¹⁴C]-NeuAc (12.0GBq/mmol) was purchased from Amersham Pharmacia Biotech. Sialidases(NANase II, III) were purchased from Glyko Inc. N-glycanase(Glycopeptidase F) was purchased from Takara Shuzo Co., Ltd. [α-³²P]dCTPwas purchased from NEN. Human Multiple tissue cDNA panel was purchasedfrom Clontech. GM1b and its positional analogs, GSC-68,2,3-sialylparagloboside (2,3-SPG), and 2,6-sialylparagloboside (2,6-SPG)were contributed from Prof. Makoto Kiso (Faculty of Agriculture, GifuUniversity). NeuAcα2,3Gal and NeuAcα2,6Gal were contributed from Dr.Hideki Ishida (The Noguchi Institute). An anti-GD3 monoclonal antibodyKM641 was contributed from Dr. Kenya Shitara and Dr. Nobuo Hanai ofKyowa Hakko Kogyo Co., Ltd. In addition, an anti-NeuAcα2,8NeuAcα2,3Galantibody S2-566 was purchased from Seikagaku Corp. Peroxidase-conjugatedAffiniPure goat anti-mouse IgG+IgM (H+L) was purchased from JacksonImmuno Research. Desialylated (asialo) glycoproteins obtained byremoving sialic acids from BSM, α1-acid glycoprotein, and ovomucoid wereprepared by treating them at 80° C. for 1 hour in 0.02 N HCl.

Using the amino acid sequence of mouse sialyltransferase ST8Sia V, aclone encoding a novel sialyltransferase showing a homology with theabove enzyme has been searched against the database of expressedsequence tag (dbEST) of the National Center for BiotechnologyInformation. As a result, clones deposited under GenBank™ accession Nos.BE633149, BE686184, and BF730564 were obtained. Based on the informationregarding the nucleotide sequences of these clones, two types ofsynthetic DNA fragments, 5′-CTTTTCTGGAGAACTAAAGG-3′ (corresponding tonucleotides 1001-1020 in FIG. 1A) (SEQ ID NO: 9) and5′-AATTGCAGTTTGAGGATTCC-3′ (corresponding to a complementary strand ofnucleotides 1232-1251 in FIG. 1A) (SEQ ID NO: 10) were prepared.Thereafter, in accordance with the method of Israel (Israel, D. I.(1993) Nucleic Acids Res. 21, 2627-2631), the cDNA library of each ofmouse brain and heart was screened by the polymerase chain reactionmethod (PCR). As a result, a clone encoding a portion of a novelsialyltransferase was obtained from each cDNA library. In order toobtain a full-length clone, two types of synthetic DNA fragments5′-TGGCTCAGGATGAGATCGGG-3′ (corresponding to nucleotides 68-87 in FIG.1A) (SEQ ID NO: 11) and 5′-TACTAGCGCTCCCTGTGATTGG-3′ (corresponding to acomplementary strand of nucleotides 725-746 in FIG. 1A) (SEQ ID NO: 12)were further prepared. Thereafter, using mouse kidney-derived cDNA as atemplate, DNA located between both the synthetic DNA fragments wasamplified by PCR. The amplified fragment was ligated to a clone obtainedfrom the mouse brain cDNA library, so as to obtain a full-length clone.This cDNA had a single open reading frame encoding type II transmembraneprotein of 398 amino acids with an estimated molecular weight of 45,399.In addition, sialyl motifs conserved in sialyltransferases were presentin the amino acid sequence thereof. This protein showed 42.0% and 38.3%homology with ST8Sia I and V, respectively, at an amino acid sequencelevel among known mouse sialyltransferases (FIG. 2A). As describedbelow, since this protein had the activity of α2,8-sialyltransferase, itwas named as the O-glycan α2,8-sialyltransferase of the presentinvention, ST8Sia VI.

On the other hand, in order to examine whether or not enzymes similar tothe above enzyme are present in other mammals, using the sequenceinformation of mouse ST8Sia VI, database was searched in the same manneras described above. As a result, it could be confirmed that similarenzymes are also present in human and rat. FIG. 1B shows the sequenceinformation of human ST8Sia VI. Mouse ST8Sia VI showed a homology of82.4% with human ST8Sia VI at an amino acid sequence level (FIG. 2B).

Subsequently, in order to examine enzymatic properties of ST8Sia VI, asecretory protein was produced. First, with regard to mouse ST8Sia VI,using two types of synthetic DNA fragments each containing a XhoI site,5′-TGCTCTCGAGCCCAGCCGACGCGCCTGCCC-3′ (corresponding to nucleotides141-170 in FIG. 1A) (SEQ ID NO: 13) and5′-TATTCTCGAGCTAAGAAACGTTAAGCCGTT-3′ (corresponding to a complementarystrand of nucleotides 1263-1293 in FIG. 1A) (SEQ ID NO: 14), a DNAfragment encoding the active domain of mouse ST8Sia VI was amplified byPCR with cloned full-length cDNA as a template. The amplified productwas cleaved with XhoI, and a cleaved portion was inserted into the XhoIsite of a mammalian expression vector, pcDSA. The obtained expressionvector was named as pcDSA-mST8Sia VI.

On the other hand, with regard to human ST8Sia VI, first, using twotypes of synthetic DNA fragments, 5′-CAATTGACATATCTGAATGAGAAGTCGCTC-3′(corresponding to nucleotides 293-315 in FIG. 1B) (SEQ ID NO: 15) and5′-TACTAACATCTCCTGTGGTTGG-3′ (corresponding to a complementary strand ofnucleotides 740-761 in FIG. 1B) (SEQ ID NO: 16), a DNA fragment wasamplified by PCR with colon adenocarcinoma CX-1-derived cDNA from HumanTumor Multiple Tissue cDNA Panels (Clontech) as a template. Thereafter,using two types of synthetic DNA fragments, 5′-CCAGTGTCCCAGCCTTTTGT-3′(corresponding to nucleotides 608-627 in FIG. 1B) (SEQ ID NO: 17) and5′-TGAGTGGGGAAGCTTTGGTC-3′ (corresponding to a complementary strand ofnucleotides 1407-1426 in FIG. 1B) (SEQ ID NO: 18), a DNA fragment wasamplified by PCR in the same manner as described above. Thereafter, thetwo amplified DNA fragments were ligated to each other, using an EcoRIsite that was common in both the amplified fragments, thereby obtaininga DNA fragment encoding the active domain of human ST8Sia VI. Thisfragment was inserted into the EcoRV site of a cloning vectorpBluescript II SK(+), and thereafter, a fragment was cleaved with MunIand XhoI. The cleaved fragment was then inserted into the EcoRI-XhoIsite of pcDSA. The obtained product was named as an expression vectorpcDSA-hST8Sia VI.

pcDSA-mST8Sia VI and pcDSA-hST8Sia VI encode a secretory fusion proteincomprising a signal peptide of mouse immunoglobulin IgM, Staphylococcusaureus protein A, and the active domain of mouse or human ST8Sia VI(which corresponds to amino acids 26-398 in the case of mouse ST8Sia VIand amino acids 68-398 in the case of human ST8Sia VI).

Using each expression vector and lipofectamine (Invitrogen), transientexpression was carried out in COS-7 cells (Kojima, N. et al. (1995) FEBSLett. 360, 1-4). The proteins of the present invention secreted from thecells into which each expression vector had been introduced were namedas PA-mST8Sia VI (mouse) and PA-hST8Sia VI (human). PA-mST8Sia VI andPA-hST8Sia VI were adsorbed to IgG-Sepharose (Amersham PharmaciaBiotech), and were then recovered from medium. Sialyltransferaseactivity was measured as follows according to the method of Lee et al.(Lee, Y.-C. et al. (1999) J. Biol. Chem. 274, 11958-11967). A reactionsolution (10 μl) containing 50 mM MES buffer (pH 6.0), 1 mM MgCl₂, 1 mMCaCl₂, 0.5% Triton CF-54, 100 μM CMP-[¹⁴C]-NeuAc, a glycoconjugate(which was added at 0.5 mg/ml in the case of glycolipids, and at 1 mg/mlin the case of glycoproteins or oligosaccharides), and a PA-mST8Sia VIor PA-hST8Sia VI suspension, was incubated at 37° C. for 3 to 20 hours.Thereafter, in the case of glycolipids, the reaction product waspurified with a C-18 column (Sep-Pak Vac 100 mg; Waters) and thepurified product was used as a sample, and in the case ofoligosaccharides or glycoproteins, the reaction product was directlyused as a sample. Thus, the obtained samples were subjected to analysis.In the case of oligosaccharides or glycolipids, the sample was spottedon a silica gel 60 HPTLC plate (Merck), and was then developed with adeveloping solvent consisting of ethanol:pyridine:n-butanol:water:aceticacid=100:10:10:30:3 (for oligosaccharides), a developing solventconsisting of 1-propanol:ammonia water:water=6:1:2.5 (foroligosaccharides), or a developing solvent consisting ofchloroform:methanol: 0.02% CaCl₂=55:45:10 (for glycolipids). In the caseof glycoproteins, analysis was carried out by SDS-polyacrylamide gelelectrophoresis. The obtained radioactivities were visualized with aBAS2000 radio image analyzer (Fuji Film) and then quantified.

Table 1 shows substrate specificity of PA-mST8Sia VI and PA-hST8Sia VI.TABLE I Acceptor substrate specificity of ST8Sia VI Using PA-mST8Sia VIand PA-hST8Sia VI, specificity against various acceptor substrates wasexamined. The concentration of the substrates is 0.5 mg/ml in the caseof glycolipids, and 1 mg/ml in the case of glycoproteins,monosaccharides and oligosaccharides. The relative activity wascalculated by taking incorporation obtained with Fetuin (PA-mST8Sia VIis 2.06 pmol/h/(ml enzyme solution), and PA-hST8Sia VI is 0.204pmol/h/(ml enzyme solution)) as 100. R represents the remainder of theN-linked sugar chain. ND: not determined Relative rate (%) Human MouseST8Sia Acceptor Representative structures of carbohydrates ST8SiaVI VIGlycoproteins 100 100 Fetuin NeuAcα2,3Galβ1,3GalNAc-O-Ser/ThrNeuAcα2,3Galβ1,3(NeuAcα2,6)GalNAc-O-Ser/Thr NeuAcα2,6(3)Galβl,4GlcNAc-RAsialofetuln 0 0 α1-Acid glycoprotein NeuAcα2,6(3)Galβ1,4GlcNAc-R 0 0Asialo- α1-Acid glycoprotein 0 0 BSM NeuAcα2,6GalNAc-O-Ser/Thr 375 24.2GlcNAcβ1,3(NeuAcα2,6)GalNAc-O-Ser/Thr Asialo-BSM 0 0 OvomucoidNeuAcα2,3Galβ1,4GlcNAc-R 6.2 12.3 Asialoovomucoid 0 0 GlycollpidsLactosylceramide Galβ1,4Glcβ1-Cer 0 ND GM4 NeuAcα2,3Galβ1-Cer 1.0 ND GM3NeuAcα2,3Galβ1,4Glcβ1-Cer 13.0 1.6 GM1aGalβ1,3GalNAcβ1,4(NeuAcα2,3)Galβ1,4Glcβ1-Cer 0 ND GD1aNeuAcα2,3Galβ1,3GalNAcβ1,4(NeuAca2,3)Galβ1,4Glcβ1-Cer 6.0 1.8 GD3NeuAcα2,8NeuAcα2,3Galβ1,4Glcβ1-Cer 0 0 GD1bGalβ1,3GalNAcβ1,4(NeuAcα2,8NeuAcα2,3)Galβ1,4Glcβ1-Cer 0 ND GT1bNeuAcα2,8Galβ1,3GalNAcβ1,4(NeuAcα2,8NeuAcα2,3)Galβ1,4Glcβ1-Cer 1.1 2.2GQ1bNeuAcα2,8NeuAcα2,8Galβ1,3GalNAcβ1,4(NeuAcα2,8NeuAcα2,3)Galβ1,4Glcβ1-Cer0 0 GM1b NeuAcα2,3Galβ1,3GalNAcβ1,4Galβ1,4Glcβ1-Cer 1.0 ND GSC-68NeuAcα2,6Galβ1,3GalNAcβ1,4Galβ1,4Glcβ1-Cer 2.6 ND 2,3-SPGNeuAcα2,3Galβ1,4GlcNAcβ1,3Galβ1,4Glcβ1-Cer 3.5 ND 2,6-SPGNeuAcα2,6Galβ1,4GlcNAcβ1,3Galβ1,4Glcβ1-Cer 0.98 ND Monosaccharides andoligosaccharides 3′-Sialyllactose NeuAcα2,3Galβ1,4Glc 629 69.96′-Sialyllactose NeuAcα2,6Galβ1,4Glc 91.5 10.73′-Sialyl-N-acetyllactosamine NeuAcα2,3Galβ1,4GlcNAc 411 ND6′-Sialyl-N-acetyllactosamine NeuAcα2,6Galβ1,4GlcNAc 88.7 ND3′-Sialylgalactose NeuAcα2,3Gal 13.9 ND 6′-Sialylgalactose NeuAcα2,6Gal2.0 ND N-Acetylneuraminic acid NeuAc 0 ND Galactose Gal 0 NDN-Acetylgalactosamine GalNAc 0 ND

PA-mST8Sia VI showed activity on glycolipids having a structure“NeuAcα2,3(6)Gal-” at the nonreducing end thereof, such as GM4, GM3,GD1a, GT1b, GM1b, GSC-68, 2,3-SPG, or 2,6-SPG. When GM3 was used as asubstrate, the incorporated sialic acid of the reaction product was notcleaved with sialidase (NANase II), which specifically cleaves α2,3- andα2,6-linked sialic acid. However, the incorporated sialic acid wascleaved with sialidase (NANase III), which specifically cleaves α2,3-,α2,6-, α2,8- and α2,9-linked sialic acids (FIG. 3A). In addition, it wasconfirmed by TLC immunostaining using an anti-GD3 monoclonal antibodyKM641 (Saito, M. et al. (2000) Biochim. Biophys. Acta 1523, 230-235)that this reaction product was GD3 into which sialic acid had beenintroduced through an α2,8-linkage (FIG. 3B). Thus, it was clarifiedthat PA-mST8Sia VI transfers sialic acid through an α2,8-linkage.

On the other hand, where a glycoprotein was used as a substrate (Table1), PA-mST8Sia showed the highest activity toward BSM, which containsonly O-glycans as glycoconjugate. PA-mST8Sia also showed activity towardFetuin, which contains both O-glycans and N-glycans and towardOvomucoid, which contains only N-glycans. However, the activity towardOvomucoid was lower than that toward a protein containing O-glycans.Moreover, PA-mST8Sia VI showed no activity on asialoglycoproteins.Furthermore, from an experiment wherein monosaccharide oroligosaccharide was used as a substrate (Table 1), it was found that theminimum sugar chain unit, which was recognized by PA-mST8Sia VI as asubstrate, is NeuAcα2,3(6)Gal.

It was found by an N-glycanase treatment that when Fetuin was used as asubstrate, the majority of sialic acid, which was newly introduced byPA-mST8Sia VI, was incorporated into O-glycans (FIG. 4). That is, whenFetuin was sialylated by PA-mST8Sia VI with [¹⁴C]-NeuAc, and thesialylated product was then treated with N-glycanase, which releasesN-glycans from a peptide portion. The majority (82.7%) of radioactivitywas still kept in the Fetuin after this treatment. This fact shows thatthe majority of sialic acid introduced by PA-mST8Sia VI was incorporatedinto O-glycans. On the other hand, the same experiment was carried outusing mouse ST8Sia III, which used N-glycans of Fetuin as substrates. Asa result, it was found that radioactivity completely disappeared.

Moreover, in order to clarify the substrate specificity and substrateselectivity of PA-mST8Sia VI, the Km and Vmax values for BSM and GM3,respectively, were obtained. With regard to BSM, the Km value was 0.03mM, the Vmax value was 23.8 pmol/h/ml enzyme solution, and the Vmax/Kmvalue was 793. With regard to GM3, the Km value was 0.5 mM, the Vmaxvalue was 0.67 pmol/h/ml enzyme solution, and the Vmax/Km value was1.34. These results show that, for PA-mST8Sia VI, O-glycans are muchmore preferable substrates than glycolipids or N-glycans.

PA-hST8Sia VI has the same enzymatic properties as those describedabove, although differences are somewhat found in activity values (Table1, and FIGS. 3A and 4). Accordingly, it can be said that ST8Sia VIderived from various types of animals had substrate specificitydifferent from that of the conventional α2,8-sialyltransferases.

In addition, concerning mouse ST8Sia VI, the in vivo enzymatic activityof the full-length clone was also examined (FIG. 5). A 1.4-kb NotI-ApaIfragment containing a region encoding the full-length mouse ST8Sia VIwas inserted into the NotI-ApaI site of an expression vector pRc/CMV,and it was named as pRc/CMV-ST8Sia VI. The vector pRc/CMV-ST8Sia VI wasintroduced into COS-7 cells using lipofectamine. Ganglioside wasextracted from the cells, and it was then subjected to TLCimmunostaining, using a monoclonal antibody S2-566 which recognizes anNeuAcα2,8NeuAcα2,3Gal structure (FIG. 5A). As a result, it was foundthat the amount of ganglioside having an NeuAcα2,8NeuAcα2,3Gal structurewas significantly increased in the cells into which pRc/CMV-ST8Sia VIhad been introduced. Moreover, with regard to glycoproteins in thecells, NeuAcα2,8NeuAcα2,3Gal structures were also newly formed onO-glycans of the cells into which pRc/CMV-ST8Sia VI had been introduced(FIG. 5B). These results show that mouse ST8Sia VI functions asα2,8-sialyltransferase in vivo.

Mouse ST8Sia VI is expressed mainly in the kidney, heart, spleen, or thelike (FIG. 6A), but human ST8Sia VI is expressed mainly in the placenta,various types of embryonic tissues, various types of tumor cells, or thelike (FIG. 6B).

Example 2 β-galactoside α2,6-sialyltransferase

The following reagents and samples were used in specific examples of thepresent invention. Fetuin, asialofetuin, bovine submaxillary mucin(BSM), α1-acid glycoprotein, ovomucoid, lactosyl ceramide (LacCer), GA1,GM3, GM1a, Galβ1,3GalNAc, Galβ1,3GlcNAc, Galβ1,4GlcNAc, Triton CF-54,and β-galactosidase (derived from bovine testis) were purchased fromSigma. Paragloboside and lactose were purchased from Wako Pure ChemicalIndustries, Ltd. CMP-[¹⁴C]-NeuAc (12.0 GBq/mmol) was purchased fromAmersham Pharmacia Biotech. Lacto-N-tetraose, Lacto-N-neotetraose, andsialidases (NANase I, II) were purchased from Glyko Inc. [α-³²P]dCTP waspurchased from NEN. Human and mouse Multiple tissue cDNA panels werepurchased from Clontech. Desialylated (asialo) glycoproteins obtained byremoving sialic acids from BSM, α1-acid glycoprotein, and ovomucoid wereprepared by treating them at 80° C. for 1 hour in 0.02 N HCl.

Using the amino acid sequence of human sialyltransferase ST6Gal I, aclone encoding a novel sialyltransferase showing a homology with theabove enzyme has been searched against the database of expressedsequence tag (dbEST) of the National Center for BiotechnologyInformation. As a result, EST clones deposited under GenBank™ accessionNos. BE613250, BE612797, and BF03852 were obtained. These clones werepurchased from the I. M. A. G. E. Consortium. Using the information ofthese nucleotide sequences, the dbEST database and the high throughputgenomic sequence database of the human genome were searched, and therelated EST clones and the genomic nucleotide sequence information ofthis gene were obtained (Accession Nos. H94068, AA514734, BF839115,AA210926, AA385852, H94143, and BF351512 (EST clones), and AC016994(genome sequence)). Based on the information on the above nucleotidesequences, primers used for the polymerase chain reaction method (PCR)were synthesized. Using these primers, PCR was performed with humancolon-derived cDNA as a template. Thereafter, the amplified fragment wasligated to the DNA fragment derived from the obtained EST clone, so asto obtain a clone containing the full-length coding region (FIG. 7A).This cDNA had a single open reading frame which encodes a type-IItransmembrane protein of 529 amino acids and it has an estimatedmolecular weight of 60,157. It was predicted from the hydropathy plotthat a transmembrane domain exists in the region corresponding to aminoacids 12-30 (FIG. 7B). The sialyl motifs conserved in sialyltransferaseswere present in the amino acid sequence of the present protein.Moreover, among the known human sialyltransferases, the present proteinshowed the highest homology (48.9%) with ST6Gal I at an amino acidsequence level (FIG. 9A), but it showed only approximately 21% to 36%homology with sialyltransferases belonging to other families. Asdescribed below, since this protein had the activity of β-galactosideα2,6-sialyltransferase, it was named as the β-galactosideα2,6-sialyltransferase of the present invention, ST6Gal II. In addition,there was a short-form clone of human ST6Gal II, having a differentsequence from the middle of sialyl motif S, which was considered to be asplicing variant (FIG. 7A).

On the other hand, in order to examine whether or not enzymes similar tothe above enzyme are present also in other mammals, database wassearched in the same manner as described above using the sequenceinformation of human ST6Gal II. As a result, it could be confirmed thatsimilar enzymes are also present in mice. Thus, cloning was also carriedout on mice. Using two types of synthetic DNA fragments,5′-GACAATGGGGATGAGTTTTTTACATCCCAG-3′ (corresponding to nucleotides321-350 in FIG. 8A) (SEQ ID NO: 19) and5′-CGATTTCCTCCCCCAAGGAGGAGTTCAGG-3′ (corresponding to a complementarystrand of nucleotides 864-893 in FIG. 8A) (SEQ ID NO: 20), a DNAfragment was amplified by PCR with mouse 14-day-old embryo-derived cDNAas a template. Moreover, using two types of synthetic DNA fragments,5′-ACGTTGGACGGCAGAGAGGCGCCCTTCTCG-3′ (corresponding to nucleotides774-803 in FIG. 8A) (SEQ ID NO: 21) and5′-ACCTTATTGCACATCAGTTCCCAAGAGTTC-3′ (corresponding to a complementarystrand of nucleotides 1582-1611 in FIG. 8A) (SEQ ID NO: 22), a DNAfragment was amplified by PCR in the same manner as described above.Thereafter, the two amplified DNA fragments were ligated to each other,using a KpnI site that was common in both the amplified fragments.Thereafter, another DNA fragment which was amplified by PCR using twotypes of synthetic DNA fragments 5′-CAATGAAACCACACTTGAAGCAATGGCGAC-3′(corresponding to nucleotides 1-30 in FIG. 8A) (SEQ ID NO: 23) and5′-CGCAACAAAAAAATAGCTATCTTCCTCGGG-3′ (corresponding to a complementarystrand of nucleotides 381-410 in FIG. 8A) (SEQ ID NO: 24), was furtherligated to the above ligated fragment, using an Aor51HI site common inboth the DNA fragments, so as to obtain a DNA fragment encoding thefull-length mouse ST6Gal II. The obtained DNA fragment was then insertedinto a cloning vector pBluescript II SK(+). FIG. 8A shows the sequenceinformation of mouse ST6Gal II. Mouse ST6Gal II consisted of 524 aminoacids, and a portion corresponding to a stem region in mouse ST6Gal IIwas approximately 5 amino acids shorter than that in human ST6Gal II. Itwas predicted from the hydropathy plot that the transmembrane domain ofthe present protein exists in a region corresponding to amino acids12-30 (FIG. 8B). Human ST6Gal II showed 77.1% homology with mouse ST6GalII at an amino acid sequence level (FIG. 9B).

Subsequently, in order to examine enzymatic properties of ST6Gal II, asecretory protein was produced. First, with regard to human ST6Gal II, aXhoI site was introduced immediately downstream of the DNA portionencoding the transmembrane domain using a synthetic DNA fragmentcontaining a XhoI site, 5′-TCATCTACTTCACCTCGAGCAACCCCGCTG-3′(corresponding to nucleotides 255-284 in FIG. 7A) (SEQ ID NO: 25). Usingthis site and a XhoI site of the pBluescript II SK(+), the XhoI fragmentencoding the stem region and active domain of ST6Gal II was prepared.This XhoI fragment was then inserted into the XhoI site of a mammalianexpression vector pcDSA. The obtained expression vector was named aspcDSA-hST6Gal II. On the other hand, with regard to mouse ST6Gal II,using a synthetic DNA fragment containing a MunI site,5′-CATCCAATTGACCAACAGCAATCCTGCGGC-3′ (corresponding to nucleotides83-112 in FIG. 8A) (SEQ ID NO: 26) instead of the synthetic DNA fragmentused in the above cloning, 5′-CAATGAAACCACACTTGAAGCAATGGCGAC-3′(corresponding to nucleotides 1-30 in FIG. 8A) (SEQ ID NO: 23), theMunI-XhoI fragment encoding the stem region and active domain of mouseST6Gal II was prepared. This fragment was then inserted into theEcoRI-XhoI site of pcDSA. The thus obtained vector was named as anexpression vector pcDSA-mST6Gal II.

pcDSA-mST6Gal II and pcDSA-hST6Gal II encode a secretory fusion proteincomprising a signal peptide of mouse immunoglobulin IgM, Staphylococcusaureus protein A, and an active domain of mouse or human ST6Gal II(which corresponds to amino acids 33-529 in the case of human ST6Gal II,and amino acids 31-524 in the case of mouse ST6Gal II).

Using each expression vector and lipofectamine (Invitrogen), transientexpression was carried out in COS-7 cells (Kojima, N. et al. (1995) FEBSLett. 360, 1-4). The proteins of the present invention secreted from thecells into which each expression vector had been introduced were namedas PA-hST6Gal II (human) and PA-mST6Gal II (mouse). PA-hST6Gal II andPA-mST6Gal II were adsorbed to IgG-Sepharose (Amersham PharmaciaBiotech), and were then recovered from medium. Sialyltransferaseactivity was measured as follows according to the method of Lee et al.(Lee, Y.-C. et al. (1999) J Biol. Chem. 274, 11958-11967). A reactionmixture (10 μl) containing 50 mM MES buffer (pH 6.0), 1 mM MgCl₂, 1 mMCaCl₂, 0.5% Triton CF-54, 100 μM CMP-[¹⁴C]-NeuAc, a substrate sugarchain (which was added at 0.5 mg/ml in the case of glycolipids, and at 1mg/ml in the case of glycoproteins or oligosaccharides), and aPA-hST6Gal II or PA-mST6Gal II suspension, was incubated at 37° C. for 3to 20 hours. Thereafter, in the case of glycolipids, the reactionproduct was purified with a C-18 column (Sep-Pak Vac 100 mg; Waters) andthe purified product was used as a sample. In the case ofoligosaccharides or glycoproteins, the reaction product was directlyused as a sample. Thus, the obtained sample was subjected to analysis.In the case of oligosaccharides or glycolipids, the sample was spottedon a silica gel 60 HPTLC plate (Merck), and it was then developed with adeveloping solvent consisting of 1-propanol:ammonia water:water=6:1:2.5(for oligosaccharides), or a developing solvent consisting ofchloroform:methanol: 0.02% CaCl₂=55:45:10 (for glycolipids). In the caseof glycoproteins, analysis was carried out by SDS-polyacrylamide gelelectrophoresis. The obtained radioactivities were visualized with aBAS2000 radio image analyzer (Fuji Film) and then quantified.

Table 2 shows substrate specificity of PA-hST6Gal II and PA-mST6Gal II.TABLE 2 Substrate specificity of ST6Gal II Using PA-hST6Gal II andPA-mST6Gal II, specificity against various substrates was examined. Theconcentration of the substrates is 0.5 mg/ml in the case of glycolipids,and 1 mg/ml in the case of glycoproteins, monosaccharides andoligosaccharides. The relative activity was calculated by taking theincorporation obtained with Gal β 1,4GlcNAc as 100. R represents theremainder of the N-linked sugar chain. Relative rate (%) Mouse HumanHuman Acceptors Representative structures of carbohydrates ST6Gal IIST6Gal II ST6Gal I Oligosaccharides Type II Galβ1,4GlcNAc 100* 1O0**100*** Type I Galβ1,3GlcNAc 0 0 4.2 Type III Galβ1,3GalNAc 0 0 0 LactoseGalβ1,4Glc 0 0 8.7 Lacto-N-tetraose Galβ1,3GlcNAcβ1,3Galβ1,4Glc 0 0 31.1Lacto-N-neotetraose Galβ1,4GlcNAcβ1,3Galβ1,4Glc 128.8 86.2 101.6Glycoproteins Fetuin NeuAcα2,3Galβ1,3GalNAc-O-Ser/Thr 0 0 13.0NeuAcα2,3Galβ1,3(NeuAcα2,6)GaINAc-O-Ser/Thr NeuAcα2,6(3)Galβ1,4GlcNAc-RAsialofetuin 21.0 3.9 95.0 BSM NeuAcα2,6GalNAc-O-Ser/Thr 0 0 0GlcNAcβ1,3(NeuAcα2,6)GalNAc-O-Ser/Thr Asialo-BSM 0 0 0 OvomucoidNeuAcα2,3Galβ1,4GlcNAc-R 0 0 9.0 Asialoovomucoid 0 0 12.7 αl-Acidglycoprotein NeuAcα2,6(3)Galβ1,4GlcNAc-R 0.75 1.2 37.1 Asialo- αl-Acidglycoprotein 12.3 1.2 93.0 Glycolipids Lactosylceramide Galβ1,4Glcβ1-Cer0 0 0 GA1 Galβ1,3GalNAcβ1,4Galβ1,4Glcβ1-Cer 0 0 0 GM1aGalβ,3GalNcβ1,4(NeuAca2,3)Galβ1,4Glcβ1-Cer 0 0 0 GM3NeuAcα2,3Galβ1,4Glcβ1-Cer 0 0 0 ParaglobosideGalβ1,4GlcNAcβ1,3Galβ1,4Glcβ1-Cer 0 0 0.3*, 2.74 pmol/h/ml medium.**, 1.03 pmol/h/ml medium.***, 8.14 pmol/h/ml medium.NeuAc,N-acetylneuraminic acid.Cer, ceramide.

Both the enzymes showed activity only on oligosaccharides having aGalβ1,4GlcNAc structure at the nonreducing end thereof (FIG. 10).Moreover, the enzymes also showed weak activity on glycoproteins, whichwere likely to have the above structure. In contrast, there were noglycolipids, which could be substrates of both the enzymes, as far asthe inventors have examined. The activity of human ST6Gal I onoligosaccharides was also examined for comparison. As a result, humanST6Gal I showed activity not only on oligosaccharides having aGalβ1,4GlcNAc structure, but also on lactose, Lacto-N-tetraose, etc.(FIG. 10). Moreover, ST6Gal I showed activity on a wide range ofglycoproteins and glycolipids (Table 2). These results show that ST6GalII has higher selectivity than ST6Gal I in terms of substratespecificity. Furthermore, it was confirmed that a short-form protein,which is a splicing variant of human ST6Gal II, had no enzyme activity(FIG. 10).

When sialic acid is transferred into Galβ1,4GlcNAc by PA-hST6Gal II orPA-mST6Gal II, as in the case of ST6Gal I, the incorporated sialic acidof the reaction product was not cleaved with sialidase (NANase I), whichspecifically cleaves α2,3-linked sialic acids. However, the incorporatedsialic acid was cleaved with sialidase (NANase II), which specificallycleaves α2,3- and α2,6-linked sialic acids (FIG. 11A). Moreover, thisreaction product showed the same mobility as that of6′-sialyl-N-acetyllactosamine in TLC, and even after the reactionproduct was treated with galactosidase, there were no changes in itsmobility in TLC (FIG. 11B). Accordingly, it was considered that thereaction product was 6′-sialyl-N-acetyllactosamine obtained byintroducing sialic acid into galactose through an α2,6-linkage. Asstated above, it was found that ST6Gal II transfers sialic acid intogalactose through an α2,6-linkage. It was considered that itsparticularly preferred substrate is an oligosaccharide having aGalβ1,4GlcNAc structure at the nonreducing end thereof.

Further, the expression patterns of human ST6Gal I and ST6Gal II invarious tissues were examined by PCR, using ST6Gal I-specific primers(5′-TTATGATTCACACCAACCTGAAG-3′ (SEQ ID NO: 27) and5′-CTTTGTACTTGTTCATGCTTAGG-3′ (SEQ ID NO: 28); the size of a PCRamplified fragment: 372 bp), and ST6Gal II-specific primers(5′-AGACGTCATTTTGGTGGCCTGGG-3′ (corresponding to nucleotides 1264-1286in FIG. 7A) (SEQ ID NO: 29) and 5′-TTAAGAGTGTGGAATGACTGG-3′(corresponding to nucleotides 1745-1765 in FIG. 7A) (SEQ ID NO: 30); thesize of a PCR amplified fragment: 502 bp) (FIG. 12A). As a result, itwas found that human ST6Gal I was expressed in almost all tissues, butthat human ST6Gal II was expressed at an extremely low level or was notexpressed at all in tissues other than the small intestine, largeintestine, or fetal brain. Moreover, it was also found that human ST6GalI was expressed in various types of tumor cells, but that the expressionof ST6Gal II was not detected in tumor cells (FIG. 12B). The expressionpattern of mouse ST6Gal II was examined in the same above manner, usingmouse ST6Gal II-specific primers (5′-CAATGAAACCACACTTGAAGCAATGGCGAC-3′(corresponding to nucleotides 1-30 in FIG. 8A) (SEQ ID NO: 23) and5′-CGCAACAAAAAAATAGCTATCTTCCTCGGG-3′ (corresponding to a complementarystrand of nucleotides 381-410 in FIG. 8A) (SEQ ID NO: 24); the size of aPCR amplified fragment: 410 bp). As a result, it was found that theexpression of mouse ST6Gal II was observed in the brain and embryo, butthat the expression thereof was observed at an extremely low level orwas not observed at all in other tissues (FIG. 12C). These resultssuggest that ST6Gal I and ST6Gal II play different roles in vivo.

INDUSTRIAL APPLICABILITY

The present invention provides a novel enzyme O-glycanα2,8-sialyltransferase, and a novel protein having an active portion ofthe enzyme and being extracellularly secreted. The enzyme and protein ofthe present invention have the activity of O-glycanα2,8-sialyltransferase. Accordingly, it is useful as a reagent forintroducing a human-type sugar chain into a protein, for example. Inaddition, the O-glycan α2,8-sialyltransferase of the present inventionis useful also as a medicament for treating hereditary diseases causedby deficiency of sugar chains specific for humans. Moreover, theO-glycan α2,8-sialyltransferase of the present invention can also beused as a medicament which acts for suppression of cancer metastasis,prevention of virus infection, suppression of inflammatory response, oractivation of neural cells. Furthermore, the O-glycanα2,8-sialyltransferase of the present invention is useful also as areagent used in studies for increasing physiological action by addingsialic acid to drugs or the like.

Still further, the present invention provides a novel enzymeβ-galactoside α2,6-sialyltransferase and a novel protein having anactive portion of the enzyme and being extracellularly secreted. Theenzyme and protein of the present invention has the activity ofβ-galactoside α2,6-sialyltransferase, and it thereby becomes possible toselectively introduce sialic acid through an α2,6-linkage into galactosesuch as oligosaccharide having a Galβ1,4GlcNAc structure. Theβ-galactoside α2,6-sialyltransferase ST6Gal II of the present inventionis useful as a therapeutic agent for treating hereditary diseases causedby deficiency of specific sugar chains synthesized by the presentenzyme, as an agent acting for suppression of cancer metastasis,prevention of virus infection, suppression of inflammatory response, oractivation of neural cells, or as a reagent used in studies forincreasing physiological action or inhibiting hydrolytic activity ofglycolytic enzymes by adding sialic acid to sugar chains.

1. O-glycan α2,8-sialyltransferase having substrate specificity andsubstrate selectivity, wherein the enzyme has substrate specificitywherein the substrates of the enzyme are glycoconjugates having aSiaα2,3(6)Gal structure wherein Sia represents sialic acid and Galrepresents galactose at the terminus thereof; and wherein the enzyme hassubstrate selectivity wherein the enzyme incorporates sialic acids intoO-glycans more preferentially than into glycolipids or N-glycans. 2.O-glycan α2,8-sialyltransferase having either one of the following aminoacid sequences: (1) an amino acid sequence shown in SEQ ID NO: 1 or 3;or (2) an amino acid sequence comprising a deletion, substitution,and/or addition of one or several amino acids with respect to the aminoacid sequence shown in SEQ ID NO: 1 or 3, and having O-glycanα2,8-sialyltransferase activity.
 3. O-glycan α2,8-sialyltransferase geneencoding the amino acid sequence of the O-glycan α2,8-sialyltransferaseaccording to claim
 2. 4. The O-glycan α2,8-sialyltransferase geneaccording to claim 3 which has any one of the following nucleotidesequences: (1) a nucleotide sequence corresponding to a portion betweennucleotide 77 and nucleotide 1270 of a nucleotide sequence shown in SEQID NO: 2; (2) a nucleotide sequence comprising a deletion, substitution,and/or addition of one or several nucleotides with respect to thenucleotide sequence corresponding to a portion between nucleotide 77 andnucleotide 1270 of the nucleotide sequence shown in SEQ ID NO: 2, andencoding a protein having O-glycan α2,8-sialyltransferase activity; (3)a nucleotide sequence corresponding to a portion between nucleotide 92and nucleotide 1285 of a nucleotide sequence shown in SEQ ID NO: 4; and(4) a nucleotide sequence comprising a deletion, substitution, and/oraddition of one or several nucleotides with respect to the nucleotidesequence corresponding to a portion between nucleotide 92 and nucleotide1285 of the nucleotide sequence shown in SEQ ID NO: 4, and encoding aprotein having O-glycan α2,8-sialyltransferase activity.
 5. Arecombinant vector comprising the O-glycan α2,8-sialyltransferase geneaccording to claim
 3. 6. The recombinant vector according to claim 5which is an expression vector.
 7. A transformant transformed with therecombinant vector according to claim
 5. 8. A method for producingO-glycan α2,8-sialyltransferase wherein the transformant of claim 7 iscultured and O-glycan α2,8-sialyltransferase is collected from theculture.
 9. A protein which comprises an active domain of O-glycanα2,8-sialyltransferase having any one of the following amino acidsequences: (1) an amino acid sequence corresponding to a portion betweenpositions 26 and 398 of the amino acid sequence shown in SEQ ID NO: 1;(2) an amino acid sequence comprising a deletion, substitution, and/oraddition of one or several amino acids with respect to the amino acidsequence corresponding to a portion between positions 26 and 398 of theamino acid sequence shown in SEQ ID NO: 1, and having O-glycanα2,8-sialyltransferase activity; (3) an amino acid sequencecorresponding to a portion between positions 68 and 398 of the aminoacid sequence shown in SEQ ID NO: 3; and (4) an amino acid sequencecomprising a deletion, substitution, and/or addition of one or severalamino acids with respect to the amino acid sequence corresponding to aportion between positions 68 and 398 of the amino acid sequence shown inSEQ ID NO: 3, and having O-glycan α2,8-sialyltransferase activity. 10.An extracellular secretory protein, comprising a polypeptide portionwhich is an active domain of the O-glycan α2,8-sialyltransferase ofclaim 1, and a signal peptide, and has O-glycan α2,8-sialyltransferaseactivity.
 11. A gene encoding the protein according to claim
 9. 12. Arecombinant vector comprising the gene according to claim
 11. 13. Therecombinant vector according to claim 12 which is an expression vector.14. A transformant transformed with the recombinant vector according toclaim
 12. 15. A method for producing a protein comprising an activedomain of O-glycan α2,8-sialyltransferase wherein the transformant ofclaim 14 is cultured and the protein is collected from the culture. 16.β-galactoside α2,6-sialyltransferase having activity and substratespecificity, wherein the activity comprises enzyme transfer of sialicacid through an α2,6 linkage into the galactose portion of a sugar chainhaving a galactose β1,4N-acetylglucosamine structure at the terminusthereof; and wherein the enzyme has substrate specificity wherein thesubstrate of the enzyme is a sugar chain having a galactoseβ1,4N-acetylglucosamine structure at the terminus thereof, and lactoseand a sugar chain having a galactose β1,3N-acetylglucosamine structureat the terminus thereof are not the substrate of the enzyme. 17.β-galactoside α2,6-sialyltransferase having either one of the followingamino acids: (1) an amino acid sequence shown in SEQ ID NO: 5 or 7; or(2) an amino acid sequence comprising a deletion, substitution, and/oraddition of one or several amino acids with respect to the amino acidsequence shown in SEQ ID NO: 5 or 7, and having β-galactosideα2,6-sialyltransferase activity.
 18. A β-galactosideα2,6-sialyltransferase gene encoding the amino acid sequence of theβ-galactoside α2,6-sialyltransferase according to claim
 17. 19. Theβ-galactoside α2,6-sialyltransferase gene according to claim 18 whichhas any one of the following nucleotide sequences: (1) a nucleotidesequence corresponding to a portion between nucleotide 176 andnucleotide 1762 of a nucleotide sequence shown in SEQ ID NO: 6; (2) anucleotide sequence comprising a deletion, substitution, and/or additionof one or several nucleotides with respect to the nucleotide sequencecorresponding to a portion between nucleotide 176 and nucleotide 1762 ofthe nucleotide sequence shown in SEQ ID NO: 6, and encoding a proteinhaving β-galactoside α2,6-sialyltransferase activity; (3) a nucleotidesequence corresponding to a portion between nucleotide 3 and nucleotide1574 of a nucleotide sequence shown in SEQ ID NO: 8; and (4) anucleotide sequence comprising a deletion, substitution, and/or additionof one or several nucleotides with respect to the nucleotide sequencecorresponding to a portion between nucleotide 3 and nucleotide 1574 ofthe nucleotide sequence shown in SEQ ID NO: 8, and encoding a proteinhaving β-galactoside α2,6-sialyltransferase activity.
 20. A recombinantvector comprising the β-galactoside α2,6-sialyltransferase geneaccording to claim
 18. 21. The recombinant vector accrding to claim 20which is an expression vector.
 22. A transformant transformed with therecombinant vector according to claim
 20. 23. A method for producingβ-galactoside α2,6-sialyltransferase wherein the transformant of claim22 is cultured and β-galactoside α2,6-sialyltransferase is collectedfrom the culture.
 24. A protein comprising an active domain ofβ-galactoside α2,6-sialyltransferase having any one of the followingamino acid sequences: (1) an amino acid sequence corresponding to aportion between positions 33 and 529 of the amino acid sequence shown inSEQ ID NO: 5; (2) an amino acid sequence comprising a deletion,substitution, and/or addition of one or several amino acids with respectto the amino acid sequence corresponding to a portion between positions33 and 529 of the amino acid sequence shown in SEQ ID NO: 5, and havingβ-galactoside α2,6-sialyltransferase activity; (3) an amino acidsequence corresponding to a portion between positions 31 and 524 of theamino acid sequence shown in SEQ ID NO: 7; and (4) an amino acidsequence comprising a deletion, substitution, and/or addition of one orseveral amino acids with respect to the amino acid sequencecorresponding to a portion between positions 31 and 524 of the aminoacid sequence shown in SEQ ID NO: 7, and having β-galactosideα2,6-sialyltransferase activity.
 25. An extracellular secretory protein,which comprises a polypeptide portion which is an active domain of theβ-galactoside α2,6-sialyltransferase according to claim 16 or 17, and asignal peptide, and has β-galactoside α2,6-sialyltransferase activity.26. A gene encoding the protein according to claim
 24. 27. A recombinantvector comprising the gene according to claim
 26. 28. The recombinantvector according to claim 27 which is an expression vector.
 29. Atransformant transformed with the recombinant vector according to claim27.
 30. A method for producing a protein comprising an active domain ofβ-galactoside α2,6-sialyltransferase wherein the transformant of claim29 is cultured and the protein is collected from the culture.